Thursday, January 31, 2013

Disinfectants are those chemicals that destroy pathogenic bacteria
from inanimate surfaces. Some chemical have very narrow spectrum of activity
and some have very wide. Those chemicals that can sterilize are called
chemisterilants. Those chemicals that can be safely applied over skin and mucus
membranes are called antiseptics. An ideal antiseptic or disinfectant should
have following properties:

• Should
have wide spectrum of activity

• Should
be able to destroy microbes within practical period of time

• Should
be active in the presence of organic matter

• Should
make effective contact and be wettable

• Should
be active in any pH

• Should
be stable

• Should
have long shelf life

• Should
be speedy

• Should
have high penetrating power

• Should
be non-toxic, non-allergenic, non-irritative or non-corrosive

• Should
not have bad odor

• Should
not leave non-volatile residue or stain

• Efficacy
should not be lost on reasonable dilution

• Should not be
expensive and must be available easily Such an ideal disinfectant is not yet
available. The level of disinfection achieved depends on contact time,
temperature, type and concentration of the active ingredient, the presence of
organic matter, the type and quantum of microbial load. The chemical
disinfectants at working concentrations rapidly lose their strength on
standing.

Application: A 70% aqueous solution is more effective at killing microbes
than absolute alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on
skin. Isopropyl alcohol is preferred to ethanol. It can also be used to
disinfect surfaces. It is used to disinfect clinical thermometers. Methyl
alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods.
Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

ALDEHYDES:

Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group,
and probably damages nucleic acids. It kills all microorganisms, including
spores.

Examples: Formaldehyde, Gluteraldehyde

Application: 40% Formaldehyde (formalin) is used for surface disinfection and
fumigation of rooms, chambers, operation theatres, biological safety cabinets,
wards, sick rooms etc. Fumigation is achieved by boiling formalin, heating
paraformaldehyde or treating formalin with potassium permanganate. It also
sterilizes bedding, furniture and books. 10% formalin with 0.5% tetraborate
sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize
thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments
etc. An exposure of at least 3 hours at alkaline pH is required for action by
gluteraldehyde. 2% formaldehyde at 40oC for 20 minutes is used to
disinfect wool and 0.25% at 60oC for six hours to disinfect animal hair
and bristles.

Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor
penetration, leaves non-volatile residue, activity is reduced in the presence
of protein. Gluteraldehyde requires alkaline pH and only those articles that
are wettable can be sterilized.

PHENOL:

Mode of
action:
Act by disruption of membranes, precipitation of proteins and inactivation of
enzymes.

Applications: Joseph Lister
used it to prevent infection of surgical wounds. Phenols are coal-tar
derivatives. They act as disinfectants at high concentration and as antiseptics
at low concentrations. They are bactericidal, fungicidal, mycobactericidal but
are inactive against spores and most viruses. They are not readily inactivated
by organic matter. The corrosive phenolics are used for disinfection of ward
floors, in discarding jars in laboratories and disinfection of bedpans.
Chlorhexidine can be used in an isopropanol solution for skin disinfection, or
as an aqueous solution for wound irrigation. It is often used as an antiseptic
hand wash. 20% Chlorhexidine gluconate solution is used for pre-operative hand
and skin preparation and for general skin disinfection. Chlorhexidine gluconate
is also mixed with quaternary ammonium compounds such as cetrimide to get
stronger and broader antimicrobial effects (eg. Savlon). Chloroxylenols are
less irritant and can be used for topical purposes and are more effective
against gram positive bacteria than gram negative bacteria. Hexachlorophene is
chlorinated diphenyl and is much less irritant. It has marked effect over gram
positive bacteria but poor effect over gram negative bacteria, mycobacteria,
fungi and viruses. Triclosan is an organic phenyl ether with good activity
against gram positive bacteria and effective to some extent against many gram
negative bacteria including Pseudomonas. It also has fair activity on fungi and
viruses.

Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is
inactivated by anionic soaps. Chloroxylenol is
inactivated by hard water.

HALOGENS: Mode of action: They are oxidizing agents and cause damage by oxidation
of essential sulfydryl groups of
enzymes. Chlorine reacts with water to form hypochlorous acid, which is
microbicidal. Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine
compounds (tincture iodine,
iodophores) Applications: Tincture of iodine (2% iodine in 70% alcohol) is an
antiseptic. Iodine can be combined with neutral carrier polymers such as
polyvinylpyrrolidone to prepare iodophores such as povidone-iodine. Iodophores
permit slow release and reduce the irritation of the antiseptic. For hand
washing iodophores are diluted in 50% alcohol. 10% Povidone Iodine is used
undiluted in pre and postoperative skin disinfection. Chlorine gas is used to
bleach water. Household bleach can be used to disinfect floors. Household
bleach used in a stock dilution of 1:10. In higher concentrations chlorine is
used to disinfect swimming pools. 0.5% sodium hypochlorite is used in serology
and virology. Used at a dilution of 1:10 in decontamination of spillage of
infectious material. Mercuric chloride is used as a disinfectant. Disadvantages: They are rapidly inactivated in the presence of organic
matter. Iodine is corrosive and staining. Bleach solution is corrosive and will
corrode stainless steel surfaces.

HEAVY METALS: Mode of action: Act by precipitation of proteins and oxidation of
sulfydryl groups. They are bacteriostatic. Examples: Mercuric chloride, silver nitrate, copper sulfate, organic
mercury salts (e.g., mercurochrome, merthiolate) Applications: 1% silver nitrate solution can be applied on eyes as
treatment for opthalmia neonatorum (Crede’s method). This procedure is no
longer followed. Silver sulphadiazine is used topically to help to prevent
colonization and infection of burn tissues. Mercurials are active against
viruses at dilution of 1:500 to 1:1000. Merthiolate at a concentration of
1:10000 is used in preservation of serum. Copper salts are used as a fungicide.
Disadvantages: Mercuric chloride is highly toxic, are readily
inactivated by organic matter.

SURFACE ACTIVE AGENTS:
Mode of actions: They have the property of concentrating at interfaces between
lipid containing membrane of bacterial cell and surrounding aqueous medium.
These compounds have long chain hydrocarbons that are fat soluble and charged
ions that are water-soluble. Since they contain both of these, they concentrate
on the surface of membranes. They disrupt membrane resulting in leakage of cell
constituents.Examples: These are soaps or detergents. Detergents can be anionic or
cationic. Detergents containing negatively charged long chain hydrocarbon are
called anionic detergents. These include soaps and bile salts. If the
fat-soluble part is made to have a positive charge by combining with a
quaternary nitrogen atom, it is called cationic detergents. Cationic detergents
are known as quaternary ammonium compounds (or quat). Cetrimide and benzalkonium
chloride act as cationic detergents. Application: They are active against vegetative cells, Mycobacteria and
enveloped viruses. They are widely used as disinfectants at dilution of 1-2%
for domestic use and in hospitals. Disadvantages: Their activity is reduced by hard water, anionic
detergents and organic matter. Pseudomonas can metabolise cetrimide, using them
as a carbon, nitrogen and energy source.

DYES: Mode of action: Acridine dyes are bactericidal because of their
interaction with bacterial nucleic acids.
Examples: Aniline dyes such as crystal violet, malachite green and
brilliant green. Acridine dyes such as acriflavin and aminacrine. Acriflavine
is a mixture of proflavine and euflavine. Only euflavine has effective antimicrobial
properties. A related dye, ethidium bromide, is also germicidal. It
intercalates between base pairs in DNA. They are more effective against gram
positive bacteria than gram negative bacteria and are more bacteriostatic in
action. Applications: They may be used topically as antiseptics to treat mild
burns. They are used as paint on the skin to treat bacterial skin infections.
The dyes are used as selective agents in certain selective media.

HYDROGEN PEROXIDE:
Mode of action: It acts on the microorganisms through its release of nascent
oxygen. Hydrogen peroxide produces hydroxyl-free radical that damages proteins
and DNA.

Application: It is used
at 6% concentration to decontaminate the instruments, equipments such as
ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and
deodorising wounds and ulcers. Strong solutions are sporicidal.

ETHYLENE OXIDE (EO):
Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl-,
amino-, carboxyl- and hydroxyl- groups. Properties: It is a cyclic molecule, which is a colorless liquid at room
temperature. It has a sweet ethereal odor, readily polymerizes and is
flammable. Application: It is a highly effective chemisterilant, capable of killing
spores rapidly. Since it is highly flammable, it is usually combined with CO2
(10% CO2+ 90% EO) or dichlorodifluoromethane. It requires presence of humidity.
It has good penetration and is well absorbed by porous material. It is used to
sterilize heat labile articles such as bedding, textiles, rubber, plastics,
syringes, disposable petri dishes, and complex apparatus like heart - lung
machine, respiratory and dental equipments. Efficiency testing is done using Bacillus
subtilis var niger. Disadvantages: It is highly toxic, irritating to eyes, skin, highly
flammable, mutagenic and carcinogenic.

BETA-PROPIOLACTONE (BPL):
Mode of action: It is an alkylating agent and acts through alkylation of
carboxyl- and hydroxyl- groups. Properties: It is a colorless liquid with pungent to slightly sweetish
smell. It is a condensation product of ketane with formaldehyde. Application: It is an effective sporicidal agent, and has broad-spectrum
activity. 0.2% is used to sterilize biological products. It is more efficient
in fumigation that formaldehyde. It is used to sterilize vaccines, tissue
grafts, surgical instruments and enzymes Disadvantages: It has poor penetrating power and is a carcinogen.

PHYSIO-CHEMICAL METHOD:
Mode of action: A physio-chemical method adopts both physical and chemical
method. Use of steam-formaldehyde is a physio-chemical method of sterilization,
which takes into account action of steam as well as that of formaldehyde.
Saturated steam at a pressure of 263 mm has a temperature of 70oC. The
air is removed from the autoclave chamber and saturated steam at
sub-atmospheric pressure is flushed in. Formaldehyde is then injected with
steam in a series of pulses, each of 5-10 minutes. The articles are held at
this holding temperature for one hour. Formaldehyde is then flushed by inflow
of steam. Disadvantages: Condensation of formaldehyde occurs and induction of
large volume of formaldehyde wets the steam resulting in loss of latent heat. Sterilization control: using paper strips containing 106 spores of G. stearothermophilus.

TESTING
OF DISINFECTANTS:

A disinfectant must be tested to know the required effective
dilution, the time taken to effect disinfection and to periodically monitor its
activity. As disinfectants are known to lose their activity on standing as well
as in the presence of organic matter, their activity must be periodically
tested.

Different
methods are:

1.Koch’s method

2.Rideal Walker Method

3.Chick Martin test

4.Capacity use dilution test (Kelsey-Sykes test)

5.In-use test

Koch’s method: Spores of Bacillus anthracis were dried on silk thread
and were subjected to action of disinfectants. Later, it was washed and
transferred to solid medium.

Rideal Walker
method:
This method relies on the estimation of phenol coefficient. Phenol coefficient
of a disinfectant is calculated by dividing the dilution of test disinfectant
by the dilution of phenol that disinfects under predetermined conditions. Both
the phenol and the test disinfectant are diluted from 1/95 to 1/115 and their
bactericidal activity is determined against Salmonella typhi suspension.
Subcultures are performed from both the test and phenol at intervals of 2.5, 5,
7.5 and 10 minutes. The plates are incubated for 48-72 hours at 37°C. That
dilution of disinfectant which disinfects the suspension in a given time is
divided by that dilution of phenol which disinfects the suspension in same time
gives its phenol coefficient.

Disadvantages of the Rideal-Walker test are: No organic matter is
included; the microorganism Salmonella typhi may not be appropriate; the time
allowed for disinfection is short; it should be used to evaluate phenolic type
disinfectants only.

Chick Martin test: This test also determines the phenol coefficient of the test
disinfectant. Unlike in Rideal Walker method where the test is carried out in
water, the disinfectants are made to act in the presence of yeast suspension
(or 3% dried human feces). Time for subculture is fixed at 30 minutes and the
organism used to test efficacy is S.typhi as well as S.aureus.
The phenol coefficient is lower than that given by Rideal Walker method.

Rideal
-Walker

Chick-Martin

Volume
medium

5.0 ml

10.0 ml

Diluent for
test disinfectant

Water

Yeast
suspension

Reaction
temperature

17.5±0.5ºC

30ºC

Organism

Salmonella
typhi

Salmonella
typhi, Staphylococcus aureus

Sampling
times

2.5, 5.0,
7.5, 10.0 min.

30.0 min.

Calculation
of coefficient

Dilution
test killing in 7.5 min divided by same for phenol

Mean
concentration of phenol showing no growth after 30 min. divided by same for
test

The classical
tests such as Rideal - Walker or Chick - Martin are not practicable.

Capacity use
dilution test (Kelsey-Sykes test):

Inoculum of four different test organisms, namely Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris
are added to the disinfectant in three successive. Dried yeast is included to
simulate presence of organic matter. The method can be carried out under
'clean' or 'dirty' conditions. The dilutions of the disinfectant are made in
hard water for clean conditions and in yeast suspension for dirty conditions.
Test organism alone or with yeast is added at 0,10 and 20 minutes interval. The
contact time of disinfectant and test organism is 8 min. The disinfectant is
evaluated on its ability to kill microorganisms or lack of it and the result is
reported as a pass or a fail and not as a coefficient. The capacity test of
Kelsey and Sykes gives a good guideline for the dilution of the preparation to
be used. Disadvantage of this test is the fact that it is rather complicated.

In-use test:

The routine monitoring of disinfectant in use can be done by the
‘in use’ test of Maurer. This test is intended to estimate the number of living
organism in a vessel of disinfectant in actual use. The disinfectant that is
already in use is diluted 1 in 10 by mixing 1 ml of the disinfectant with 9 ml
of sterile nutrient broth. Ten drops of the diluted disinfectant (each 0.02 ml)
is placed on two nutrient agar plates. One plate is incubated at 37oC for 3
days while the other is held at room temperature for 7 days. The number of
drops that yielded growth is counted after incubation. If there growth in more
than five drops on either plate, it represents failure of disinfectant.